Methods for isolating nucleic acids are well known from the state of the art. Accordingly, according to one of the most established methods in the state of the art, DNA is isolated from biological starting materials—such as cells and tissues—by solubilising the starting materials containing the nucleic acid under strongly denaturising and reducing conditions (partially also by using protein-degrading enzymes) and isolating the thus released nucleic acids from the aqueous phase by the dialysis method or by means of an ethanol precipitation [J. Sambrock, E. F. Fritsch and T. Maniatis, 1989, Cold Spring Harbor, “Molecular Cloning”].
The main disadvantage of this method is seen in the fact that the isolation of nucleic acids from cells and in particular from tissues has shown itself to be very time-consuming and can frequently take more than two days. In addition, this method necessitates a considerable outlay on apparatus, and includes the use of substances such as phenol or chloroform, which irritate the skin or damage health.
In view of this situation, alternative methods were developed at an early stage in the state of the art, which are intended to enable the disadvantages involved in the extraction of nucleic acids described above to be avoided.
All these methods are based on a method developed and described for the first time by Vogelstein and Gillespie (Proc. Natl. Acad. Sci, USA, 1979, 76, 615-619) for the preparative and analytical cleaning of DNA fragments from agarose gels. The method combines the dissolving of the agarose containing the DNA band to be isolated in a saturated solution of a chaotropic salt (NaJ) with a bonding of the DNA to glass particles. The DNA fixed to the glass particles is subsequently washed with a washing solution (20 mM Tris HCl [pH 7.2], 200 mM NaCl; 2 mM EDTA; 50% v/v ethanol) and then desorbed from the carrier particles.
This methodology has undergone a series of modifications up to today and at the present time is used for different methods of extracting and cleaning nucleic acids of different provenances (Marko, M. A., Chipperfield, R. and Birnboim, H. G., Anal. Biochem., 121, (1982) 382-387).
Accordingly, numerous reagent combinations (so-called kits) are commercially available for carrying out nucleic acid extractions of this kind.
These almost exclusively commercially available kits are based on the sufficiently well-known principle of bonding nucleic acids to mineral carriers under the presence of solutions of different chaotropic salts, and use suspensions of finely ground glass powder (e.g. Glasmilk, BIO 101, La Jolla, Calif.), diatomic earths (Sigma) or even silica gels (European patent application 616 639) as carrier materials.
A method of isolating nucleic acids, which in principle is practical for a large number of different applications, has been disclosed in Boom et al. [EP 389 063 A1]. In this European patent application, a method is described for isolating nucleic acids from starting materials containing nucleic acids by incubating the starting material with a chaotropic buffer of a DNA-bonding solid phase. The chaotropic buffers realise both the lysis of the starting material and also the bonding of the nucleic acids to the solid phase. The method is also suitable for isolating nucleic acids from small sample quantities, and finds practical use particularly in the field of isolating viral nucleic acids.
Although problems, which develop due to a possibly difficult lysis of the starting material, can be solved by a series of commercially available products for the isolation of nucleic acids (especially for the isolation of genomic DNA from complex starting materials), they conceal however the major disadvantage that this is no longer a classic “single tube method”, which characterises the above method according to the disclosure of Boom et al., as the lysis of the starting material is carried out in an ordinary buffer using a proteolytic enzyme. The chaotropic ions necessary for the subsequent bonding of the nucleic acids to centrifugation membranes, for example, must be additionally added to the lysis preparation when the lysing is complete. In no circumstances can they form part of the lysis buffer, as the protein-destroying function of chaotropic salts is well known and would naturally immediately destroy the proteolytic enzyme necessary for efficient lysis.
In spite of a series of disadvantages, the methods of nucleic acid isolation using chaotropic salts have become established worldwide and are applied in their millions using commercially available products. These systems are extremely simple in their execution and in all cases proceed in accordance with the following principle:                Lysis of the starting material, subsequent bonding of the nucleic acids to the solid phase of a glass or silica membrane, which is located on a carrier suspension in a centrifuge column;        Washing of the bonded nucleic acids, and        Elution of the nucleic acids with a buffer of low ion strength;        
All these systems are based on the bonding of the nucleic acids to the respective carrier surfaces in the presence of chaotropic salts, i.e. at least one buffer solution contains a chaotropic salt as its main component. Under certain circumstances, this can even apply to the lysis buffer or—in systems that use proteolytic enzymes—to a necessary bonding buffer, which is added to the starting material after lysis is complete.
The Hofmeister series for salting out negatively charged, neutral or basic protein solutions forms the basis for the selection of suitable chaotropic salts. Chaotropic salts are characterised by denaturing proteins, increasing the solubility of unpolar substances in water, and destroying hydrophobic interactions. According to the state of the art, just these characteristics also effect the destruction of the superimposed structure of the aqueous environment with buffer systems of chaotropic salts, in order to promote the bonding of the nucleic acids to selected solid phases. The best-known agents for isolating nucleic acids are sodium perchlorate, sodium iodide, potassium iodide, guanidinium-isothiocyanate and guanidinium hydrochloride. However, they are cost-intensive on the one hand and to some extent toxic or irritant on the other.
The physical-chemical principle of the bonding of nucleic acids to mineral carriers in the presence of chaotropic salts has been explained in professional circles. The bonding of nucleic acids to the surfaces of mineral carriers consists in the breaking down of superimposed structures of the aqueous environment, by means of which the nucleic acids adsorb on the surface of mineral materials, in particular of glass or silica particles. The presence of chaotropic ions is always necessary to break down the superimposed structures of the aqueous environment. When the concentrations of the chaotropic salts are high, the reaction proceeds almost quantitatively. As a result of this physical-chemical knowledge that has been described, it is assumed in the state of the art that all commercially available systems for isolating nucleic acids must contain buffer compositions with high ion strengths of chaotropic salts for bonding nucleic acids to a nucleic-acid-bonding solid phase.
In addition, appropriate methods are also known from the state of the art, which manage without the use of chaotropic substances or without the use of phenol, by means of which the disadvantages described above can be avoided. International patent application WO 00/034463 discloses methods of this kind for isolating nucleic acids from complex starting materials. In these methods, a lysing/bonding buffer system is used, which has at least one antichaotropic salt component and works with a so-called alcoholic bonding chemistry (Invisorb® Plasmid Kit produced by Invitek, Berlin).
The use of alcohols, however, also has some disadvantages, which in particular include the following:                Exactly determined volume ratios of sample lysis to alcohol must be maintained meticulously accurately for plasmid isolation in order to prevent contamination of the plasmid to be isolated—in particular with RNA.        Both the chaotropic and the alcoholic bonding chemistry result in DNA isolates, which are strongly contaminated with endotoxins. Additional washing steps are limited to the use of buffers containing large amounts of alcohol in order to be able to maintain the bond to the solid phase—usually a membrane. Selective washing steps, which make the use of other substances necessary, can therefore hardly be used for this purpose.        The endotoxin content in the isolated DNA can lead to problems with the further use of the DNA isolated in this manner—for example in pharmaceutical applications.        